Helicobacter pylori is a gram negative bacterium which colonizes the gastric epithelium of up to half of the world's population and plays a causative role in the development of gastric and duodenal ulcers and gastric adenocarcinoma. One of the hallmarks of H. pylori is its persistence, and bacteria are not cleared by the host immune system. This may be explained in part by the fact that H. pylori is readily phagocytosed by macrophages, but the internalized bacteria are not killed. Significantly, preliminary data obtained by the PI suggest the following hypothesis; H. Pylori survives for at least 20 hours inside macrophages by disrupting phagosome maturation. Moreover, this appears to occur by a novel mechanism that involves 1) delayed phagocytosis 2) homotypic fusion of early phagosomes and 3) bacteria-stimulated secretion of lysosomal enzymes from infected cells. The long-term objective of this study is to dissect the mechanism of H. pylori survival in macrophages at the molecular level and to identify the host and bacterial factors required for this process. Specifically, the PI will characterize the H. pylori phagosome in macrophages and use immunofluorescence and confocal microscopy to quantify phagosome pH; electron microscopy to determine phagosome structure; and video imaging of live cells to determine whether H. pylori phagosomes interact with the endosomal compartment. Subcellular fractionation and Western blotting, and immuno-electron microscopy, and antisense oligonucleotides will be used to define the roles of phosphatidylinositol 3-kinase, protein kinase C-zeta, and rab5 in phagocytosis of H. pylori. In addition, whether macrophage-activating cytokines and/or serum opsonins increase phagocytic killing of H. pylori will be determined. Finally, H. pylori mutants with known mutations in urease and VacA will be used to assess whether these bacterial factors are essential for bacterial survival inside macrophages. These data may be the first indication that H. pylori can disrupt phagosome maturation in macrophages. A complete dissection of this process at the molecular level may lead to novel therapies for treatment of H. pylori infection and reduce the significant morbidity associated with ulcer disease.